Humans are constantly exposed to various mixtures, such as tobacco smoke, auto exhaust, and other environmental pollutants, containing several thousand compounds, including many known carcinogens. Covalent binding of reactive metabolites to DNA and proteins with the formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal effective dose and are an integral part of science-based risk assessment. Technical limitations, however, have prevented comprehensive assessment of a board spectrum of different adducts simultaneously. Consequently, most studies have focused on determination of abundant individual or a select few adducts. These studies have produced valuable insights into metabolism of individual carcinogens. Unfortunately they are insufficient in providing accurate and comprehensive data needed for assessment of the risk posed by exposure to mixtures. Thus, our long-term goal is to overcome this limitation by developing a sensitive and specific method for quantitative profiling of a broad spectrum of reactive compounds or their metabolites using hemoglobin adducts as surrogate biomarkers. We recently established an immunoaffinity liquid chromatography-tandem mass spectrometry method (LC-MS/MS) consisting of trypsin hydrolysis and sample enrichment by an adduct-specific immunoaffinity chromatography (IAC) prior to quantitation by LC-MS/MS. We propose herein to redesign the IAC enrichment procedure to enable simultaneous quantitation of a broad spectrum of N-terminal valine adducts. We base this redesign on the hypothesis that a broad spectrum of alkylated N-terminal peptides can be enriched with the use of antibodies raised specifically against the C-terminus of the target peptide. Our preliminary studies suggest that this new design is suitable for bio-monitoring. To test this hypothesis, we will (Aim 1) demonstrate proof-of-principle of the proposed multi-adduct-monitoring method with alkylated peptide standards and globin treated in vitro with alkylating agents known to form N-terminal valine adducts;and (Aim 2) establish the suitability of the multi-adduct-monitoring method for in vivo bio-monitoring using globin from mice and rats exposed to various alkylating agents at concentrations lower than experienced during smoking and at levels of occupational settings. We propose to establish this methodology for mice, rats, and humans to enable translational research. This novel redesign enabling determination of the internal doses of several carcinogens simultaneously will make it possible to (a) better understand the behavior of individual compounds in mixtures, and (b) generate more comprehensive exposure data needed for accurate risk assessment of exposure to mixtures. The proposed methodology can easily be extended to include additional adducts of interest and adapted to investigate alkylation at sites other than the N-terminal valine, providing a general analysis approach to understanding how reactive agents interact with macromolecules to exhibit their adverse effects. PUBLIC HEALTH RELEVANCE: Humans are constantly exposed to various mixtures, such as tobacco smoke, auto exhaust, and other environmental pollutants, containing several thousand compounds, including many known carcinogens. The causal link between exposure to genotoxic mixtures and the development of adverse health effects is believed to be the binding of carcinogens or their metabolites to DNA and proteins forming so called adducts. Technical limitations, however, have prevented the assessment of a multiple adducts simultaneously. Therefore, we propose to develop a method to make it possible to determine the presence and number of multiple adducts simultaneously in order to obtain accurate information needed to assess the risks to human health posed by exposure to mixtures.